Beta-globin recombinant lentiviral vector and application thereof

A technology of recombinant lentivirus and globin, which is applied in the field of molecular biology, can solve the problems of high production cost of vectors and low titer of lentivirus packaging, and achieve the effects of reducing potential carcinogenic risk, low production cost and high packaging titer

Active Publication Date: 2022-05-06
GENMEDICN BIOPHARMA INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Technical problem to be solved: In view of the above technical problems, the present invention provides a β-globin recombinant lentiviral vector

Method used

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  • Beta-globin recombinant lentiviral vector and application thereof
  • Beta-globin recombinant lentiviral vector and application thereof
  • Beta-globin recombinant lentiviral vector and application thereof

Examples

Experimental program
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Embodiment 1

[0038] Embodiment 1: vector construction

[0039]1. Construction of lentiviral vector pCCL-SIN-cPPT-LCR2.7K-β-globin-Genomic-T87Q-RbPA, including vector pCCL-SIN-cPPT-MCS-RbPA (SEQ ID No: 1), 2.7kb β- LCR regulatory sequence (SEQ ID No:2), β-globin promoter sequence (SEQ ID No:3), genomic sequence encoding β-globin (SEQ ID No:4), β-globin polyA sequence (SEQ ID No: 5) and β-globin enhancer sequence (SEQ ID No: 6).

[0040] 1.1 Digest the pBSK vector plasmid with restriction endonucleases ClaI and XhoI at 36.5-37.5°C for 0.8-1.2h, and cut the gel to recover the pBSK vector fragment after agarose electrophoresis;

[0041] will express Hbβ A-T87Q The genome sequence of the expression cassette is divided into two parts, β-globin1 and β-globin2, and PCR is carried out respectively, wherein the primer sequence PCR1 used for the β-globin1 gene fragment is SEQ ID No: 7, PCR2 is SEQ ID No: 8, and the β-globin2 gene The primer sequence PCR3 used for the fragment is SEQ ID No: 9, and ...

Embodiment 2

[0075] Example 2: Production and purification of lentivirus

[0076]2.1 Inoculate the successfully constructed pCCL-SIN-cPPT-LCR2.7K-β-globin-Genomic-T87Q-RbPA plasmid and lentiviral packaging kit plasmid Mix in a ratio of 1:3 in a 10-layer cell factory the day before transfection For HEK293T cells, the fresh DMEM medium was replaced 6 hours after transfection, and the supernatant was collected 72 hours later for chromatographic purification;

[0077] 2.2 Purification adopts tangential flow filtration-chromatography system, uses core700 chromatography, Q ImpRes chromatography purification process, and purifies lentivirus. The specific purification process is as follows:

[0078] ① Benzonase treatment (nuclease digestion): Treat with 25U / mL Benzonase at 37°C for 1 hour to remove residual plasmid DNA and genome released from lysed cells during transfection;

[0079] ② MF (clarification): The virus harvest solution is filtered (0.45 μm) to remove insoluble particles such as HEK2...

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Abstract

The invention discloses a beta-globin recombinant lentiviral vector and application of the beta-globin recombinant lentiviral vector, the beta-globin recombinant lentiviral vector is pCCL-SIN-cPPT-LCR < 2.7 > K-beta-globin-Genomic-T87Q-RbPA, the beta-globin recombinant lentiviral vector comprises a pCCL-SIN-cPPT-MCS-RbPA skeleton, a 2.7 kb beta-LCR regulatory sequence and an expression cassette for expressing beta globin Hb beta A-T87Q with mutant amino acid at the 87th position, and the beta-LCR is a abbreviation of beta-globin chloride regulation. The lentiviral vector disclosed by the invention is high in packaging titer and low in vector production cost, the potential carcinogenic risk of the viral vector on tested cells is reduced, the Hb betaA-T87Q is expressed more efficiently and stably, and an important basis is provided for curing blood transfusion dependent type beta-thalassemia patients.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a β-globin recombinant lentiviral vector and its application. Background technique [0002] β-thalassemia is one of the most common monogenic autosomal recessive genetic diseases in the world. It is a fatal and disabling blood disease that seriously threatens human health. High incidence in West Asian (2-5%), Southeast Asian (up to 10%), and South Asian (up to 18%) populations. In my country, the high-incidence areas are mainly distributed in the provinces and regions south of the Yangtze River, among which the Guangdong and Guangxi regions are the most serious, and with the increase of population mobility, it has shown a tendency to spread to the north. There are currently an estimated 200,000 beta-thalassemia patients receiving treatment worldwide. [0003] Currently, the widely implemented clinical treatment methods for β-thalassemia include standardize...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/12C12N7/01A61K48/00A61K38/42A61P7/06
CPCC12N15/86C12N7/00C07K14/805A61K48/005A61K38/42A61P7/06C12N2740/15021C12N2740/15043
Inventor 董文吉张艳君刘子瑾董祖伊赵忠亮程谟斌
Owner GENMEDICN BIOPHARMA INC
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